Attentional Processing in C57BL/6J Mice Exposed to Developmental Vitamin D Deficiency

Lauren R. Harms1, Karly M. Turner1, Darryl W. Eyles1,2, Jared W. Young3, John J. McGrath1,2,4, Thomas H. J. Burne1,2* 1 Queensland Brain Institute, The University of Queensland, St Lucia, Queensland, Australia, 2 Queensland Centre for Mental Health Research, The Park Centre for Mental Health, Richlands, Queensland, Australia, 3 Department of Psychiatry, University of California San Diego, La Jolla, California, United Sates of America, 4 Discipline of Psychiatry, The University of Queensland, St Lucia, Queensland, Australia

Abstract Epidemiological evidence suggests that Developmental Vitamin D (DVD) deficiency is associated with an increased risk of schizophrenia. DVD deficiency in mice is associated with altered behaviour, however there has been no detailed investigation of cognitive behaviours in DVD-deficient mice. The aim of this study was to determine the effect of DVD deficiency on a range of cognitive tasks assessing attentional processing in C57BL/6J mice. DVD deficiency was established by feeding female C57BL/6J mice a vitamin D-deficient diet from four weeks of age. After six weeks on the diet, vitamin D- deficient and control females were mated with vitamin D-normal males and upon birth of the pups, all dams were returned to a diet containing vitamin D. The adult offspring were tested on a range of cognitive behavioural tests, including the five- choice serial reaction task (5C-SRT) and five-choice continuous performance test (5C-CPT), as well as latent inhibition using a fear conditioning paradigm. DVD deficiency was not associated with altered attentional performance on the 5C-SRT. In the 5C-CPT DVD-deficient male mice exhibited an impairment in inhibiting repetitive responses by making more perseverative responses, with no changes in premature or false alarm responding. DVD deficiency did not affect the acquisition or retention of cued fear conditioning, nor did it affect the expression of latent inhibition using a fear conditioning paradigm. DVD-deficient mice exhibited no major impairments in any of the cognitive domains tested. However, impairments in perseverative responding in DVD-deficient mice may indicate that these animals have specific alterations in systems governing compulsive or reward-seeking behaviour.

Citation: Harms LR, Turner KM, Eyles DW, Young JW, McGrath JJ, et al. (2012) Attentional Processing in C57BL/6J Mice Exposed to Developmental Vitamin D Deficiency. PLoS ONE 7(4): e35896. doi:10.1371/journal.pone.0035896 Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan Received February 13, 2012; Accepted March 23, 2012; Published April 26, 2012 Copyright: ß 2012 Harms et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Project Grant no. 511066 from the National Health and Medical Research Council of Australia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts. TB is a PLoS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. The authors have declared that no other competing interests exist. * E-mail: [email protected]

Introduction but hyper-exploration has not been observed in the rat model [8]. We have also shown that DVD deficiency altered brain An increasing body of epidemiological evidence supports the morphology in C57BL6/J, but not 129X1/SvJ mice, in which hypothesis that maternal vitamin D deficiency is a risk-modifying adult C57BL/6J male DVD-deficient mice had smaller lateral factor for schizophrenia [1,2]. In a recent case-control study, those ventricles compared to controls, which may have been compressed with low levels of vitamin D at birth had a two-fold increase in the by the enlarged striatum seen in these DVD-deficient mice [10]. risk of developing schizophrenia in later life [3]. Moreover, there is This is in contrast to what is seen in DVD-deficient rats, which now convincing experimental evidence based on a model in have increased lateral ventricular volume [11,12]. In addition, Sprague-Dawley rats that developmental vitamin D (DVD) DVD-deficient rats were found to be hypersensitive to the deficiency is associated with a range of schizophrenia-related locomotor-stimulating effects of the psychotomimetics d-amphet- neurochemical, neuroanatomical, and behavioural phenotypes amine and MK-801, indicating possible disruptions in the [4,5]. As such, these data support the biological plausibility of the dopamine and glutamate systems of these animals [9,13,14,15]. epidemiological link between DVD deficiency and schizophrenia. Such effects were not seen in DVD-deficient mice [10]. Another To date, most of the animal experimental work related to DVD study also found that DVD-deficient C57BL/6J mice have subtly deficiency has been performed in rat models. More recently, two impaired performance in an olfactory tubing maze, which is an strains of mice (C57BL/6J and 129X1/SvJ) have been used to operant conditioning paradigm requiring mice to learn the examine the effect of DVD deficiency on adult mouse behaviour associations between a scent and reward [16]. These findings and it was found that in both strains, DVD deficiency promoted a support the hypothesis that there are DVD deficiency-induced hyper-explorative phenotype [6]. Furthermore, DVD deficiency effects on behaviourally-relevant neuronal systems. However, little was associated with enhanced spontaneous locomotion in 129X1/ is known regarding how DVD deficiency affects cognitive SvJ, but not C57BL/6J mice [6]. Spontaneous hyperlocomotion behaviours that are specifically relevant to schizophrenia. has also been observed in DVD-deficient rats several times [7,8,9],

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Cognitive impairments associated with schizophrenia are possible candidate for models combining both genetic and predictive of functional outcomes [17,18]. Generally, these environmental factors. Therefore, the aim of the current study impairments are not responsive to antipsychotic drugs [19]. was to further examine learning, memory and cognition in DVD- Attention/vigilance is one major cognitive function that is deficient mice by investigating attention/vigilance using the 5C- impaired in patients with schizophrenia [20,21]. Attention is SRT and 5C-CPT, and learning, memory and LI using a fear typically measured in patients using the Continuous Performance conditioning paradigm. Test (CPT), and a version of this (CPT-Identical Pairs, IP) has been recommended for use in assessing attention in patients with Results schizophrenia [22]. The CPT can assess attentional performance by measuring a patient’s ability to respond to target stimuli, while Attention/Vigilance withholding their response to non-target (or noise) stimuli. Using 5C-SRT. Both control and DVD-deficient mice had similar signal detection theory, several parameters can be used to assess learning rates in the 5C-SRT training levels (F1,34 = 2.03, performance in the CPT. One such example is the discrimination P = 0.163; Fig. 1). All mice required the maximum number of index (d9) that can be used to grade the subject’s ability to testing sessions to reach criteria for level 7 (stimulus discriminate between targets and non-targets. duration = 1 s). On the 3-day multiple stimulus durations level, Several tests have now been developed in rodents with the aim mice were challenged to respond to a range of stimulus durations of modelling the tests in humans, such as the CPT, which can be (0.2–1.0 s). There was no effect of Maternal Diet on any measure used to examine attention. The 5-Choice Serial Reaction Task of performance in the multiple stimulus durations task, including (5C-SRT) was developed as a test of attention in rats [23]. It has accuracy (F1,34 = 3.11, P = 0.087; Fig. 2a), omission rate become one of the primary methods used to assess aspects of (F1,34 = 1.20, P = 0.281; Fig. 2c), premature responses attentional function in rodents, yet this test does not truly (F1,34 = 0.38, P = 0.540; Fig. 2e) or perseverative responses recapitulate the human test (CPT) that is widely used to measure (F1,34 = 1.67, P = 0.205; Fig. 2g). At low stimulus durations, all attention. This is because there are no non-target trials in the 5C- mice made more omissions (F1,34 = 69.78, P,0.001; Fig. 2c) and SRT in which the animal is expected to withhold their response. were less accurate when responding (F1,34 = 235.04, P,0.001; Therefore, to more accurately model the human CPT, Young et Fig. 2a). On the 3-day multiple inter-trial intervals level, there was al. (2009) developed the Five-Choice Continuous Performance no effect of Maternal Diet on performance over various inter-trial Test (5C-CPT) in mice, which was comprised of target and non- intervals on measures such as accuracy (F1,34 = 0.04, P = 0.837; target trials. Target trials progress in a similar fashion to trials in Fig. 2b), omission rate (F1,34 = 2.59, P = 0.117; Fig. 2d), premature the 5C-SRT, requiring a response to a singly lit aperture, but in responses (F1,34 = 0.11, P = 0.737; Fig. 2f) or perseverative non-target trials, the whole array of five lights were lit and the responses (F1,34 = 0.32, P = 0.575; Fig. 2g). Over the different mouse was required to withhold a nose poke response to obtain a inter-trial intervals, the omission rate was fairly stable, except for reward. This method allows the quantification of the parameters an increase in omissions in trials after an inter-trial interval of such as d9, thus effectively modelling the tests that are used to 1.0 s, (Effect of inter-trial interval F4,34 = 97.6, P,0.002; Fig. 2d). assess the attentional impairments seen in patients with schizo- 5C-CPT. Over the 20-session 5C-CPT test, DVD-deficient phrenia. mice had similar levels of performance to controls on the primary Another aspect of attention that is altered in psychiatric measures of performance; P(Hit) (F1,34 = 2.01, P = 0.165; Fig. 3a), disorders, including schizophrenia, is Latent Inhibition (LI). LI is P(FA) (F1,34 = 1.57, P = 0.219; Fig. 3b), and d9 (F1,34 = 0.05, a learning phenomenon in which pre-exposure to an inconse- P = 0.830; Fig. 3c). DVD-deficient mice appeared to make more quential stimulus diminishes future learning of new associations for that stimulus [24]. Patients with schizophrenia, as well as several animal models of risk factors for schizophrenia, exhibit disrupted LI, meaning that pre-exposure to a stimulus does not affect leaning new associations for that same stimulus [25]. LI can be measured in rodents in many conditioning paradigms, including fear conditioning and active avoidance. Disruptions in LI were found in DVD-deficient rats [26]. Control rats that were preexposed (PE) to the conditioned stimulus (CS) before training in an active avoidance test exhibited fewer avoidance responses during training compared to controls in the non-preexposed (NPE) group, indicating that they had not learnt the association as well as the NPE group and therefore that LI had occurred in the control population. However, PE DVD-deficient rats performed equally to NPE DVD-deficient rats, indicating that pre-exposure to the CS did not impair subsequent learning (i.e. disrupted LI) [26]. Although DVD-deficient rats have selective cognitive impair- ments [26], DVD-deficient mice have thus far only been tested in a limited array of cognitive tasks [6,16] and were found to have subtly impaired performance compared to controls. However, none of the investigations performed in DVD-deficient mice to date directly assessed any of the domains of cognition that are Figure 1. Acquisition of the 5C-SRT. The graphs show the number of sessions (days) to reach criteria for control (n = 19) and DVD-deficient impaired in schizophrenia. It is important to examine the effects of (n = 19) mice on each of the increasingly-challenging training levels DVD deficiency on cognition in a mouse model because of the (with stimulus duration decreasing from 30 to 1 s). Data are expressed wealth of models of genetic risk factors for schizophrenia available as mean 6 SEM. in C57BL/6 mice, making the mouse model of DVD deficiency a doi:10.1371/journal.pone.0035896.g001

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Figure 2. Performance of mice on the 5C-SRT with multiple stimulus durations (left) and multiple inter-trial intervals (right). This figure shows accuracy (A and B), the omission rate (C and D), premature responses (E and F) and perseverative responses (G and H) for control (n = 19) and DVD-deficient (n = 19) mice over the five different stimulus durations (0.2–1.0 s) and over the five different inter-trial intervals (1–9 s). Data are expressed as mean 6 SEM. doi:10.1371/journal.pone.0035896.g002

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Figure 4. Perseverative responding in the 5C-CPT. This figure Figure 3. Performance of mice on the 5C-CPT. This figure shows shows the perseverative correct responses (A and B)andthe the P(Hit) (A), the P(FA) (B), the discrimination index d9 (C) and perseverative false alarm responses (C and D) for control (n = 9–10) premature responses (D) for control (n = 19) and DVD-deficient (n = 19) and DVD-deficient (n = 9–10) males (left) and females (right) averaged mice averaged over 4 blocks of 5 trials. DVD-deficient mice also made over 4 blocks of 5 trials. DVD-deficient males made more perseverative more premature responses than control mice, although this did not responses during target trials, particularly in the later sessions (Sessions reach significance (Main effect of Diet F1,34 = 3.66, P = 0.064). Data are 6–10 t1,17 = 22.32, P = 0.033; Sessions 11–15 t1,17 = 23.16, P = 0.006; expressed as mean 6 SEM. * P,0.05 and t P,0.1 between control and Sessions 16–20 t1,17 = 22.31, P = 0.034). Data are expressed as mean 6 DVD-deficient mice. SEM. * P,0.05 and t P,0.1 between control and DVD-deficient mice. doi:10.1371/journal.pone.0035896.g003 doi:10.1371/journal.pone.0035896.g004

testing period (effect of time F1,58 = 17.95, P,0.01; Fig. 5b). On premature responses than control mice, although this did not day 4 of testing, all mice displayed retention of conditioned fear as reach significance (Main effect of Diet F = 3.66, P = 0.064; shown by the increase in freezing responses upon CS onset (effect 1,34 , Fig. 3d). of Time F1,58 = 65.69, P 0.05; Fig. 5c), but maternal diet was not associated with an alteration in the expression of conditioned fear DVD-deficient males made more perseverative responses for the CS (F1,58 = 0.59, P = 0.446). during target trials (Sex6Diet interaction F1,34 = 6.42, P = 0.016; Fig. 4a), particularly in the later sessions (Sessions 6–10 Latent Inhibition t1,17 = 22.32, P = 0.033; Sessions 11–15 t1,17 = 23.16, P = 0.006; LI was examined on day 4 of testing, and was evident as a Sessions 16–20 t1,17 = 22.31, P = 0.034). This effect on persever- ative responses was restricted to the correct responses during target decrease in conditioned fear in PE mice (F1,114 = 13.03, P,0.001; trials, not the false alarm responses in non-target trials, for which Fig. 6). Post-hoc t tests indicated that LI was present in both control (t = 2.69, P = 0.009) and DVD-deficient mice there was no significant effect of Maternal Diet (F1,34 = 2.50, 1,59 P = 0.123; Fig. 4c,d). (t1,59 = 2.59, P = 0.012). Furthermore, DVD-deficient mice did not exhibit an altered LI response, as there was no significant Fear conditioning Maternal Diet6Pre-Exposure interaction (F1,114 = 0.01, P To exclusively assess fear conditioning in DVD-deficient mice, a = 0.907). separate analysis of mice in the NPE group was performed. There was no effect of Maternal Diet on the acquisition of the association Nociception between the CS and the US, because DVD-deficient mice The hot plate and tail flick tests revealed that DVD-deficient exhibited similar levels of conditioned freezing to control mice mice were similar to control mice in their latencies to remove their paws/tail from painful stimuli. In the hot plate test, control mice on day two of testing (F1,58 = 0.24, P = 0.628; Fig. 5a). All mice exhibited an increase in CS-evoked freezing throughout the had a latency of 18.9960.46 s to remove their paws from the heat source and DVD-deficient mice a latency of 18.1760.59 s session (effect of CS number F1,58 = 55.18, P,0.05). On day 3 of testing, DVD deficiency was not associated with a (F1,118 = 1.33, P = 0.251). For the tail flick test, mice had latencies of 4.0960.13 s and 4.0360.12 s to move their tails for the control change in the extinction of contextual fear (F1,58 = 0.10, P = 0.757). All groups exhibited the predicted decrease in freezing over the and DVD-deficient groups, respectively (F1,118 = 0.33, P = 0.565).

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Figure 5. Acquisition, contextual fear extinction and cued test of conditioned fear. Data show the freezing scores (%) over the 10630 s CS presentations in the acquisition session (Day 2; A), the 363 min sampling intervals in the context extinction session (Day 3; B) and for the 3 min interval prior to CS onset, followed by the 661 min time bins after CS onset in the retention test session (Day 4; C) for control (n = 31) and DVD- deficient (n = 31) mice in the NPE group. Data are expressed as mean 6 SEM. doi:10.1371/journal.pone.0035896.g005

Discussion there was a predictable effect of inter-trial interval on premature responses, whereby all mice made more premature responses at This study presents the first characterization of the consequenc- longer inter-trial intervals. es of DVD deficiency on attentional processing in adult mice. We In order to investigate aspects of vigilance and selective attention found that DVD deficiency in mice was associated with subtle in DVD-deficient mice, the 5C-CPT was used. In the 5C-CPT alterations in performance on the 5C-CPT, independent from any experiments, both DVD-deficient and control mice began the changes in overall performance in the domains of attention, sessions with their d9 level at approximately 0, indicating that mice learning, memory and LI. were not discriminating between the target and non-target trials. Responses on the 5C-SRT revealed that sustained and selective Throughout the sessions, however, the d9 improved in all groups attention in mice was largely unaltered by DVD deficiency when and by session 20, was at least 0.50 in all groups, indicating that the mice were presented with only target stimuli. Both control and mice were indeed responding appropriately in both target and DVD-deficient mice learned the 5C-SRT and progressed through non-target trials. DVD-deficient mice exhibited similar P(Hit) and the training levels at similar rates. Moreover, when challenged P(FA) levels to control mice throughout the 20 sessions and had with manipulations in which the stimulus duration and inter-trial similar d9 levels, indicating that discrimination was not affected by interval were varied, DVD-deficient mice performed similarly to DVD deficiency when the fundamental measures of signal controls. At low stimulus durations and with increased attentional detection were investigated. load, all mice made more omissions, and when they did respond, There were, however, some subtle effects of DVD deficiency on had lower accuracy, consistent with previously published findings performance in the 5C-CPT. DVD-deficient male mice made in the 5C-SRT [27,28]. In the inter-trial interval manipulations more perseverative responses in the CPT, but not during the early sessions (1–5). In target trials, a perseverative response occurred when a mouse had already made a correct nose poke and continued to make repeated nose pokes before collecting their reward. In non-target trials, perseverative responses occurred after a mouse had made a false alarm response and made more nose pokes during the time out period. Therefore, perseverative responses on target and non-target trials represent slightly different responses: one is the repetition of a correct response that leads to reward, and the other is the repetition of an incorrect response that leads to a time out or punishment. By definition, perseverative responses occur after the mouse has made the response that ends the trial (either with a reward or a time out) and therefore are not rewarded or punished. Increased perseverative responses may indicate that DVD- deficient mice have a response inhibition deficit. However, DVD- deficient mice had normal response inhibition in other elements of the task; they did not make more false alarms, although there was a non-significant trend towards an increase in the amount of Figure 6. LI in the retention test session (Day 4). Data show the premature responses in DVD-deficient mice. Although both freezing scores (%) for control and DVD-deficient mice in the NPE perseverative and premature responses represent a lack of (n = 31) and PE (n = 30) groups. Mice in the PE groups exhibit less inhibition, there are several differences between the two. conditioned freezing than NPE mice (Main effect of Pre-exposure Premature responses represent motoric impulsivity, or the inability F1,114 = 13.03, P,0.001), indicating LI (the inhibition of learning caused by pre-exposure to the CS). Data are expressed as mean 6 SEM. to wait and to withhold a planned response. On the other hand, * P,0.05 between NPE and PE mice. perseverative responses may represent compulsive behaviour, or doi:10.1371/journal.pone.0035896.g006 the inability to inhibit the repetition of actions. Further evidence

PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35896 Attentional Processing in C57BL/6J Mice suggests that impairments in inhibition with regard to premature has already been found that DVD deficiency in mice did not affect (impulsive) responses are neurobiologically separate from persev- levels of social exploration in the social interaction test, nor did it erative (compulsive) responses [29]. There is some evidence to affect open arm exploration on the elevated plus maze [6], suggest that premature and false alarm responses are governed by indicating that the DVD-deficient mouse, while perhaps repre- separate systems [30] and the current study adds to this body of senting a model of OCD- and autism-like repetitive behaviours, knowledge by further divorcing perseverative correct responses may not model other aspects of autism and OCD, such as social from false alarm and perseverative false alarm responses, interaction impairments and anxiety-like behaviour. There is, indicating that the 5C-CPT is a sensitive test that can be used to however, some emerging evidence that DVD deficiency may also discriminate between very specific types of response inhibition. be a risk factor for autism. There are as yet no epidemiological This is an important consideration for animal models of studies demonstrating a direct association between autistic neuropsychiatric disorders because of the shift towards investigat- spectrum disorders and developmental vitamin D levels, yet there ing individual endophenotypes of disorders (e.g. compulsivity or are effects of season of birth, latitude of birth, and migrant status impulsivity) as opposed to entire disorders (e.g. attention deficit that may indicate a possible association (for review, [39]). If further hyperactivity disorder or obsessive-compulsive disorder) [31]. evidence continues to support this association, the DVD-deficient The changes in perseverative responding, if indicative of a mouse model may be utilized to further examine the role vitamin phenotype of repetitive or compulsive behaviour, could be D plays in brain development with relevance to autism. interpreted within the context of previous findings from studies The findings from the remainder of the study indicate that in DVD-deficient mice, in which C57BL/6J mice exposed to DVD deficiency in mice was not associated with alterations in fear DVD deficiency were hyper-explorative on the hole board test, conditioning, LI or nociception. LI was demonstrated in both head dipping more over the two-day test than their control control and DVD-deficient mice, where the PE group expressed counterparts. This behaviour may indicate a hyper-responsiveness less conditioned freezing during the CS than mice in the NPE to rewarding stimuli. After initial neophobia, exploration of a group in the testing session. Disruptions in LI were previously novel environment is highly rewarding for a rodent [32]. In the found in DVD-deficient rats [26], in conjunction with several 5C-CPT, DVD-deficient mice only display increased responses other behavioural and neuroanatomical alterations relevant to that they have learnt to be associated with a reward (perseverative schizophrenia [8,9,11,12,13,14], which have not been observed in correct responses). Taken together, previous findings of enhanced the mouse model [6,10,16]. Species differences in the response to exploration and those here of a perseverative reward-associated DVD deficiency have been previously reported in regards to the response, may suggest a reward-seeking phenotype in these novel effects on exploratory behaviour [6], which were not seen in animals. Further study could be aimed at probing the function DVD-deficient rats [8]. Furthermore, whereas we have reported of the reward system in these animals, perhaps by investigating increased ventricular volume in DVD-deficient rats [11,12] the free-choice drug seeking behaviours or by examining place- DVD-deficient mouse has a reduction in ventricular volume. preference for drugs of abuse. However, such a reward-seeking These differences may not necessarily be due to species differences, phenotype would typically be associated with other behaviours but may be due to the strain selection. DVD deficiency has been that have not been observed in DVD-deficient C57BL6/J mice, examined in one outbred rat strain and two inbred mouse strains, namely, spontaneous hyperlocomotion and hypersensitivity to and perhaps the two strains chosen (C57BL/6J and 129X1/SvJ) amphetamine [32]. may be largely unresponsive to changes in vitamin D levels. Such An alternative explanation for the increased head dipping and differences highlight the importance of species and strain selection perseverative responding seen in DVD-deficient mice may be that in studies of altered vitamin D signalling and brain development. these behaviours are not related to reward, but may reflect the Taken together these findings indicate that attentional process- repetitive and compulsive behaviours commonly associated with ing is largely intact in a C57Bl/6 mouse model of prenatal vitamin obsessive-compulsive disorder (OCD) and autism. Such an D deficiency, suggesting that DVD deficiency has little to no effect association is strengthened by the possible link between the on the systems governing attention in the mouse. Nevertheless, behavioural changes identified in the current study, and utilizing a mouse model of DVD-deficiency leaves open the morphological changes found in previous studies. Firstly, both possibility to combine the model of an environmental risk factor the behavioural and morphological alterations were only identified for schizophrenia with one of the numerous models of genetic risk in male, not female mice [10,16]. Secondly, the neuroanatomical factors available in C57Bl/6 mice. Perhaps such gene6environ- changes seen in DVD-deficient mice have been associated with ment models will reveal that a combination of factors results in repetitive behaviour in clinical populations. A reduction in the cognitive impairments associated with schizophrenia. lateral ventricles was identified in DVD-deficient male mice [10,16], which has also been found in patients with autism [33]. Materials and Methods This reduction in ventricular volume in DVD-deficient mice may have been due to an enlargement of the striatum [10]. Not only Ethics Statement have increases in caudate volume been identified in populations All procedures were performed with the approval of The with autism [34], but increases in the volume of the right caudate University of Queensland Animal Ethics Committee, under the nucleus were associated with the severity of repetitive, OCD-like guidelines of the National Health and Medical Research Council behaviours in patients [35]. This is also in agreement with of Australia. evidence that the size of the striatum is increased in patients with OCD [36,37]. The changes in striatal volume seen in DVD- Animals and Housing deficient males may therefore indicate a mechanism underpinning A total of 160 C57BL/6J mice were used for the study. Mice the increases in repetitive behaviour seen in these mice. Further were housed in individually ventilated OptiMice cages (Animal exploration of this phenotype could be directed at assaying other Care Systems, CO, USA), at a stable temperature of 2161uC. For repetitive behaviours in the model, such as barbering, marble the 5C-SRT and 5C-CPT experiments, 19 males (nine control burying or reversal learning (the range of OCD-associated and 10 DVD-deficient) and 19 females (10 control and nine DVD- behaviours and models is reviewed elsewhere [38]). However, it deficient) were used from approximately 20 weeks of age. The

PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35896 Attentional Processing in C57BL/6J Mice remainder of the mice were used in the fear conditioning, LI and 5C-SRT. Mice were trained to perform the 5C-SRT nociception experiments. These mice were divided into two groups according to a standard protocol [40]. Each mouse was trained – PE and NPE, resulting in n values of 20–22 per maternal diet for or tested on one session per day for 5–7 days/week. To start each males and 9–11 per maternal diet for females. The discrepancy in testing session, the reward was raised into the reward magazine sample sizes between males and females was due to a low number and the reward light was left on. When the mouse head dipped of female mice being born at those particular times. However, on into the reward magazine to drink the reinforcer, the session average, the breeding program generated similar amounts of male began. In the first training level (0), lights did not appear in the and female offspring. Mice were reared, housed and tested at the nose poke holes and mice needed to poke into any hole to retrieve Queensland Brain Institute animal facility, The University of their reward, which was signalled by the reward light. This trial Queensland. Unless otherwise indicated (for the 5C-SRT and 5C- was run for 30 min. Mice needed to receive 70 rewards in one CPT), mice had free access to food and water throughout testing. session to proceed to the next phase of testing. DVD-deficient and control offspring were generated as For the next level of testing (level 1), mice had to respond to a previously described [6]. Briefly, female mice were fed on a diet light randomly presented in one of five holes with a nose poke in free of vitamin D (Vitamin D Deficient AIN93G Rodent diet, the lit hole to receive a reward. These were target trials, where the Specialty Feeds, WA, Australia) from 4 weeks of age. Control mouse had to respond to receive a reward. For all training phases, animals were housed in the same conditions as vitamin D-deficient the time between each trial (the inter-trial interval) was set at 5 s animals, but were given a standard vitamin D-containing diet and the stimulus duration began at 30 s for level 1. If a mouse did (Standard AIN93G Rodent diet, Specialty Feeds, WA, Australia). not respond correctly during the stimulus duration, this was scored At 10 weeks of age, all females were mated with vitamin D normal as an ‘‘omission’’ and the mouse received a ‘‘time out’’ (i.e. house males by pairing one male with 2 females for 7 days. Dams light on, no stimulus lights, no reward) for 5 s before the remained on these respective diets throughout gestation. Pregnant commencement of the next trial. The mouse also received a time dams were checked every 12 h for pups and the food of dams on out if they nose poked into an unlit hole (incorrect response), or if the vitamin D-deplete diet was replaced with control food within they nose poked during the inter-trial interval (premature 12 h of birth. Offspring were weaned 21 days after birth, and were response). If a mouse made more than one nose poke response provided with water and standard vitamin D containing mouse in a correct hole, this was counted as a perseverative response and pellets (Feeder and Grower diet, Specialty Feeds, WA, Australia) was also recorded. To reach criteria for the next level of training, a for the remainder of the experiment. After weaning, control and mouse needed to perform ,20% omissions and have an accuracy DVD-deficient offspring were re-identified to conceal the .80% (accuracy was calculated as the percentage of correct experimental group and all behavioural testing and data responses/total number of responses). Training levels 2–7 processing were performed blind to maternal diet. proceeded in much the same way as level 1, with the stimulus duration decreasing to 1 s in level 7. The time the mouse had to Attention/Vigilance make a response also decreased with the stimulus duration, but Food Restriction. Beginning from 19–21 weeks of age, one remained at 5 s in the last training levels, even when the stimulus week before the commencement of training, mice were placed on was only present for 1 s. a restricted feeding schedule to decrease body weight to ,90% of Once mice had passed criteria for level 7 on 3 days, they were their original free-feeding weight. Mice were weighed, then each tested for their response to multiple stimulus durations (level 8) in box of mice was given 0.075 g of standard feed/g of starting which the stimulus duration varied randomly at values of 0.2, 0.4, weight/mouse/day at approximately 1600 h. Mice were usually 0.6, 0.8 and 1 s (all previous trials were 1 s). All mice were tested housed in pairs unless their weight loss was uneven, suggesting that for 3 days on multiple stimulus durations regardless of their one mouse was eating the majority of the food. In these cases, mice performance. After another two sessions of baseline (level 7), mice were separated until their weight had stabilized at 90%. After 5 were then tested for 3 days for their response to multiple inter-trial days of food restriction, the daily amount of food was increased to intervals (level 9), which varied randomly at values of 1, 3, 5, 7 and 0.1 g food/g free-feeding body weight. Mice were weighed each 9 s. Their baseline responses were tested again for another 2 days day prior to testing and food amounts were titrated based on their on level 7. weight, so they remained at ,90% free-feeding weight without 5C-CPT. After the last 2 days of level 7 (after multiple inter- falling lower than 85%. Mice had free access to water in the home trial intervals), sessions were altered so that the number of trials cage throughout testing. On the 5 days of food restriction 1 ml of was increased to 120 and the inter-trial intervals were variable at liquid reinforcer (Breaka strawberry milk, Parmalat, QLD, 3, 4, 5, 6 and 7 s at level 10. Once a mouse had passed criteria for Australia) was provided to each box of two mice to habituate level 10 (omissions ,25%, accuracy .80%), they were advanced them to the reinforcer. to the 5C-CPT (level 11). In the 5C-CPT, 80 of the 120 trials were Testing Apparatus. All training and testing took place in a target trials and were the same as level 10 (with a variable inter- bank of four nine-hole nose poke mouse operant chambers (Med trial interval). The other 40 trials were non-target trials and in Associates, VT, USA). However, only five of the nine holes were used in experiments (the first, third, fifth, seventh and ninth holes). order to receive a reward, the mouse needed to withhold a Each nine-hole chamber was placed within a larger sound response. For non-target trials, all nine lights were turned on for attenuated cabinet (inner dimensions 60635656 cm high) so 1 s, and the mouse had to withhold a nose poke response for 3 s in that sound and light exposure could be minimized in the testing order to receive a reward. If the mouse made a nose poke response environment. The chambers were controlled by a central interface within the 3 s, this was counted as a ‘‘false alarm’’ and they device, which was connected to a computer running MED-PC IV. received a 5 s time out. In a similar fashion to the 5C-SRT, nose MED-PC IV was used to run MedState notation programming pokes during the inter-trial interval were punished with a time out. (.mpc files), which can be used to define and initiate experiment In addition to the perseverative responses after a correct response protocols. For all experiments, modified versions of the .mpc file in a go trial, perseverative false alarms were also recorded. Mice for a standard nine-choice serial reaction time task supplied with were tested for 20 consecutive days on the 5C-CPT regardless of the system were used. performance.

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Dependent Variables. Learning rates in the operant 5C- generator to deliver the CS. A fluorescent light (PHM-258-X) was SRT and 5C-CPT tasks were examined by comparing the number in the top of the chamber, and was left on throughout of sessions it took the mice to reach criteria for each level. The experiments. The whole chamber sat within a larger sound dependent variables for these tests and how they were calculated attenuating cubicle (inner dimensions 60670640 cm high), which are outlined in Table 1. The sessions the 5C-SRT were exclusively had a small video camera attached to the door (VDO-CAM-4.65). comprised of target trials in which accuracy, P(Hit), omission rate, When the door was closed, the chamber (and the mouse inside) number of premature responses, number of perseverative could be viewed using the camera, the output of which was used responses and the latencies to make a correct response, an by the Med Associates software, Video Freeze. The software used incorrect response and to collect reward were used to analyse the the video file to calculate the motion index, the number of pixels performance of mice. Dependent variables from the multiple that had changed within a designated time period more than they stimulus durations level were averaged over the three testing would change if the mouse was not present (i.e., video noise) [41]. sessions for each mouse and pooled based on stimulus duration. Freezing was measured every 2 s and anything under a motion The same procedure was performed for dependent variables from threshold of 20 arbitrary units was regarded as freezing (10–20 is the multiple inter-trial intervals level, which were pooled based on recommended). For each time bin tested, Video Freeze returned a inter-trial interval. percentage freezing value, which was the percentage of the One-third of the trials for the 5C-CPT study, were non-target number of freezing episodes out of the total number of freezing trials, and therefore for this study, the variables P(FA), number of episodes possible in the time bin (30/min). Chambers were perseverative false alarms, latency to make a false alarm response cleaned with 70% ethanol after each test. and latency to retrieve a reward for a correctly withheld response The LI training and testing protocol was performed similarly to were added to the analysis with the other variables for target trials. that described in [42]. On the first day of testing, mice were placed Furthermore, the P(Hit) of target trials and the P(FA) of non-target in the chamber for an initial 5 min habituation period. For mice in trials were also used to calculate the discrimination index, d9. the PE group, the program for day one consisted of two 3-min sessions without any stimuli at the beginning and end, with 40610 s presentations of a 9 KHz tone, which were pseudo- Fear Conditioning and LI randomly timed, so that the time between tones was no less than Mice were tested over 4 consecutive days (one test/day) using a 30 s and no more than 90 s. The program ran for 52 min. NPE Med Associates (VT, USA) fear conditioning system (Med-VFC- mice were put in the chambers, but received no stimulus MS). Four Med Associates fear conditioning chambers (ENV-008 presentations over the 52 min trial. FC) were used, which had inner dimensions of 30625620 cm From the second day of testing, the programs for PE and NPE high. Each of the chambers had a grid floor (ENV-005 QD) mice were identical. Mice were placed in chambers and left to attached to an electric shock stimulator/scrambler (ENV-414S) to habituate for 5 min before the program and recording began. deliver the US and a speaker attached to a programmable tone Again, the first 3 min of the program were stimulus-free, after

Table 1. Descriptions of the dependent variables used for the analysis of the 5C-SRT and 5C-CPT and how they were calculated.

Measure (Target Trials)

Accuracy No. of correct responses No. correct responses+no. incorrect responses P(Hit) No. of correct responses Probability of a hit response Total no. of target trials Omission Rate No. of omissions Total no. of target trials Premature Responses No. of premature responses made Perseverative Responses (Target trials) No. of perseverative correct responses made during target trials A perseverative response occurred when a mouse nose-poked more than once in the correct hole Latency to Correct Response (s) Average time between target stimulus onset and correct nose poke Latency to Incorrect Response (s) Average time between target stimulus onset and incorrect nose poke Latency to Retrieve Reward (s) Average time between correct nose poke and reward retrieval

Measure (Non-target Trials)

P(FA) No. of false alarm responses Probability of a false alarm response Total no. of non-target trials Perseverative False Alarm (non-target trials) No. of perseverative false alarm responses made during non-target trials In contrast to perseverative responses in target trials, a perseverative response during a non-target trial occurred when a mouse made more than one false alarm nose poke Latency to False Alarm (s) Average time between non-target stimulus onset and false alarm nose poke d9 Z score of P(Hit) – Z score of P(FA) Discrimination Index

doi:10.1371/journal.pone.0035896.t001

PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35896 Attentional Processing in C57BL/6J Mice which, 10630 s 9 KHz tones (CS) were presented 1 min apart, tube (Falcon, BD, NJ, USA) for restraint purposes, with breathing each immediately followed by a 1 s duration 0.4 mA foot shock holes drilled into the end and the mouse’s tail and hind legs (US). After the CS/US pairings, recording continued for another protruding. An automated tail flick apparatus (Harvard Instru- 3 min stimulus-free period. The current protocol did not use ments, MA, USA) was used and a high-intensity light was focused different contexts for the training and test periods. Therefore, to on the dorsal surface of the tail, approximately 15 mm from the minimize contextual fear conditioning, mice were placed in the tip. An infrared beam was used to determine when the mouse’s tail chamber for 18 min without any tone or shock presentations on moved and the latency for the tail to move was recorded for three the third day of testing. consecutive trials for each mouse. Mice were removed from the On day 4, mice were placed in the chamber for 12 min. After light source if they had not moved their tails after 10 s. 3 min of baseline, stimulus-free recording, the CS (9 KHz tone) was played continuously for 6 min and followed by another 3 min Statistical Analyses baseline recording period. The dependent variables used for All data were analysed using the SPSS software package (ver. analysis of fear conditioning and LI were percentage freezing levels 17, SPSS Inc., IL, USA). For the 5C-SRT and 5C-CPT, variables during CS presentation on the acquisition day (day 2), throughout that were expressed as a fraction or rate (i.e. accuracy, omission the trial to extinguish contextual fear (day 3) and before and rate, p(Hit) and p(FA) were subjected to arcsine transformation to during the CS test on day 4 in just the NPE mice, in order to assess limit the effect of an artificially-imposed ceiling [43]. For all tests, learning and memory independent of pre-exposure. LI was ANOVA were used to assess effects of Maternal diet, Sex and Pre- examined by comparing the freezing levels in NPE and PE on exposure (in the case of LI experiments), on the main dependent the CS test day (day 4). variables for each experiment. Repeated measures factors were used where appropriate (e.g. CS number for LI experiment, Nociception stimulus duration for multiple stimulus durations experiment). Approximately 5 days after the final day of testing, all mice Significant main effects were followed by post hoc t-tests. tested in the fear conditioning/LI experiments were tested for their Significance was set at P,0.05. Where no significant sex responses to painful stimuli using the hot plate and tail flick tests. A interactions were observed data were presented pooled for sex. standard hot plate (Harvard Instruments, MA, USA) was heated to 55uC for 10 min prior to testing, and a tall Perspex cylinder was placed on the plate to contain the mouse. Mice were timed from Author Contributions the moment its back feet first touched the plate until one or both Conceived and designed the experiments: LH KT JY TB. Performed the feet left the plate (either from the mouse licking or jumping). Mice experiments: LH. Analyzed the data: LH TB. Contributed reagents/ were removed from the plate if they had not lifted their feet after materials/analysis tools: DE JM TB. Wrote the paper: LH DE JM KT JY 30 s. For the tail-flick test, mice were placed head-first into a 50 ml TB.

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